The present program is concerned with 1) the structure and diversity of function of proteolytic enzymes of various homologous classes; 2) the biological events which are initiated by protease action; and 3) evolutionary relationships among certain proteases and proteins. Particular attention will be given to trypsin-like enzymes involved in fertilization and in blood clotting, to carboxypeptidases of yeast, spleen or plasma and to the neutral metalloendopepidases of bacterial origin. Processes of limited proteolysis, as exemplified by zymogen activation, fertilization and blood clotting will be explored in detail. Evolutionary relationships will be probed by sequence analysis of various proteolytic enzymes, Factor X of the blood clotting system, rabbit and dogfish phosphorylases and other proteins. In these investigations, the following experimental methods will be of particular importance: Automated sequence analysis (sequenator), affinity chromatography, active site labelling with site-specific reagents, enzyme kinetics and reversible enzyme inhibition, and x-ray crystallography.